To maintain genomic integrity, eukaryotic cells duplicate their DNA precisely once before each cell division. In the G1 phase, pre-replication complexes (pre-RCs) are assembled at origins via the sequential recruitment of ORC, Cdc6, Cdt1, and MCM2-7. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is strictly prohibited. As a result, only one initiation event occurs at each origin, and DNA replication is limited to a single round. In vertebrates, pre-RC assembly is blocked in S phase due to Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, this laboratory made the surprising discovery that the destruction of Cdt1 in S phase is intimately coupled to DNA replication. Cdt1 is ubiquitylated on chromatin by the E3 ubiquitin ligase Cul4-Ddb1Cdt2, and this ubiquitylation event requires the interaction of Cdt1 with the processivity factor PCNA at the DNA replication fork. Studies from other laboratories suggest that PCNA-dependent Cdt1 destruction is conserved in humans, flies, worms, and possibly fission yeast. This pathway represents a new paradigm in which temporal control of proteolysis involves activation of a substrate (Cdt1) via its docking onto a cell-cycle regulated structure (chromatin-bound PCNA). This proposal contains experiments that will elucidate the mechanism by which PCNA-dependent Cdt1 destruction is temporally controlled. In Specific Aim 1, we address how Cdt1 interacts selectively with chromatin-bound PCNA to insure that Cdt1 is normally never destroyed in G1. In Specific Aim 2, we characterize the degron motif of Cdt1 and examine how it mediates binding to PCNA and Cul4-Ddb1Cdt2. In Specific Aim 3, we reconstitute Cdt1 ubiquitylation in a purified system so that we may distinguish between different mechanisms for how PCNA stimulates this process. Finally, in Specific Aim 4, we use real-time fluorescence microscopy to address how rapidly Cdt1 is destroyed at S phase onset, and we test a new model for local control of proteolysis by the PCNA/Cul4-Ddb1Cdt2 pathway. Together, these studies will illuminate the molecular mechanism underlying a novel proteolysis pathway that maintains genome stability. Narrative To maintain the integrity of our genomes and prevent diseases such as cancer, it is crucial that cells make one precise copy of their DNA before each cell division. This laboratory has discovered a new mechanism, which insures the accuracy of genome duplication in all higher organisms, and it involves the destruction of a DNA replication factor called Cdt1 after the first round of chromosome duplication has been initiated. The work is highly relevant for human health because failure to destroy Cdt1 is correlated with human cancer, and has been shown to cause cancer in mice.